Method development for metabolites of CWA-related compounds in mussel and fish samples Hanna Niemikoski VERIFIN/ Hanna Niemikoski 31.5.2019

Aim of the study Method development for metabolites of CWAs from fish and mussel samples: Synthesis of reference chemicals for metabolites of arsenic containing CWA’s and sulfur mustard Synthesis of isotope labelled internal standards Sample preparation for mussel tissue, cod tissues and bile/urine Optimization of LC-MS conditions for target analytes Sample analysis VERIFIN/ Hanna Niemikoski 31.5.2019

Timeline VERIFIN/ Hanna Niemikoski 31.5.2019

Possible metabolites of arsenic related CWA’s in fish and mussel Mussels and fish have GST activity [1,2] → glutathione (GSH) conjugates GSH conjugates are hydrolyzed further → cysteine and N-acetylcysteine conjugates are formed (more stable species) Arsenic binds to cysteine residues of proteins → arsenic-protein complex can be isolated and after enzymatic digestion arsenic-peptide complex is possible to analyze VERIFIN/ Hanna Niemikoski 31.5.2019

An example of in vitro experiment with plasma and CLARK I Plasma was exposed to CLARK I After isolation of proteins and enzymatic digestion CLARK-cysteine adduct was detected Mobile phase: 0,1 % FA in H2O and 0,1 % FA in MeOH Stationary phase: C18 Ionisation: ESI+ MRM transitions: m/z 350 → m/z 278 m/z 350 →m/z 229 VERIFIN/ Hanna Niemikoski 31.5.2019

Hypothesis: Possible metabolic pathway for glutathione conjugation of CLARK I VERIFIN/ Hanna Niemikoski 31.5.2019

Sample pretreatment method Target metabolites of DA and DM for screening of mussel and fish samples Metabolite Category Reference chemical Matrix Sample pretreatment method LC-MS/MS method As-GSH Possible metabolite of CLARK I and Adamsite x Fish (bile, urine, liver) Mussel (DG)  Under development As-Cys As-NAC* Fish (bile, urine,liver) As-protein complex Protein adduct of CLARK I X** Fish and mussel tissues Under development *N-acetylcysteine ** Analyzed as cysteine conjugate of CLARK I after enzymatic digestion VERIFIN/ Hanna Niemikoski 31.5.2019

Sulfur mustard metabolism Sulfur mustard forms protein and DNA adducts and glutathione conjugates [3,4] VERIFIN/ Hanna Niemikoski 31.5.2019

Sample pretreatment method Target metabolites of sulfur mustard for screening of mussel and fish samples Metabolite Category Reference chemical Matrix Sample pretreatment method LC-MS/MS method S-HETE-CysProPhe  Albumin adduct x  Fish blood S-HETE-Val  Haemoglobin adduct O6-HETEG DNA adduct Fish blood, urine   S-HETE-GSH glutathione metabolite FIsh urine/bile SBMSE  β-lyase metabolite  Fish urine MSMTESE SBMTE VERIFIN/ Hanna Niemikoski 31.5.2019

DNA adduct of sulfur mustard LC-MS/MS spectra Mobile phase: 0,1 % FA in H2O and 0,1 % FA in MeOH Stationary phase: C18 Ionisation: ESI+ Collision energy: 10 V VERIFIN/ Hanna Niemikoski 31.5.2019

Possible challenges Study of metabolism is essential, there is no information which metabolites are formed in fish and mussel Complex sample matrix Stability of metabolites GSH derivatized adducts Protein and DNA adducts VERIFIN/ Hanna Niemikoski 31.5.2019

References 1. Hellou, J. Environ. Sci. Pollut. Res. Int. 2012, 19, 2007-23. 2. Blanchette, B.; Feng, X.; Singh, B. R. Marine biotechnology. 2007, 9, 513-542. 3. Xu, H.; Nie, Z.; Zhang, Y.; Li, C.; Yue, L.; Yang, W.; Chen, J.; Dong, Y.; Liu, Q.; Lin, Y. Toxicology Reports. 2014, 1, 533-543. 4. Halme, M.; Pesonen, M.; Hakala, U.; Pasanen, M.; Vähäkangas, K.; Vanninen, P. Rapid Communications in Mass Spectrometry. 2015, 29, 1279-1287. VERIFIN/ Hanna Niemikoski 31.5.2019

Thank you for your attention! VERIFIN/ Hanna Niemikoski 31.5.2019